SPAC15A10.06 Antibody

What is SPAC15A10.06 protein and what cellular functions does it mediate?

SPAC15A10.06 is an uncharacterized Na(+)/H(+) antiporter found in Schizosaccharomyces pombe (fission yeast). It functions as a multi-pass membrane protein localized to the Golgi apparatus membrane. The protein belongs to the Monovalent cation:proton antiporter 1 (CPA1) transporter (TC 2.A.36) family. As a membrane transporter, it likely plays a critical role in maintaining ion homeostasis within the Golgi apparatus, potentially regulating pH and cation concentrations crucial for proper protein modification and trafficking.

Current research indicates that SPAC15A10.06 may participate in:

• Golgi ion homeostasis

• Protein processing and trafficking

• Cellular stress responses involving ion flux

• Membrane potential regulation in secretory pathways

The protein has been assigned UniProt number O13726 and Entrez Gene ID 2542747 .

What types of SPAC15A10.06 antibodies are currently available for research?

Current commercially available SPAC15A10.06 antibodies include:

The antibody is unconjugated (primary), with rabbit IgG isotype, and is raised against recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPAC15A10.06 protein .

What are the optimal protocols for Western blot detection using SPAC15A10.06 antibody?

When designing Western blot experiments with SPAC15A10.06 antibody, researchers should consider the following protocol optimizations:

• Sample Preparation:

  • For membrane proteins like SPAC15A10.06, use membrane-specific extraction buffers containing 1-2% non-ionic detergents (Triton X-100 or NP-40)

  • Maintain samples at 4°C during extraction to prevent protein degradation

  • Include protease inhibitors to preserve protein integrity

• Gel Electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal separation

  • Load 20-50μg of total protein per well

  • Include molecular weight markers spanning 25-150 kDa range (SPAC15A10.06 expected MW can be verified through UniProt data)

• Transfer and Detection:

  • For membrane proteins, extended transfer times (90-120 minutes) at lower voltage may improve efficiency

  • Block with 5% non-fat milk or BSA in TBST

  • Dilute SPAC15A10.06 antibody 1:500 to 1:2000, optimizing based on signal intensity

  • Incubate overnight at 4°C for maximum sensitivity

  • Use appropriate controls (pre-immune serum as negative control, recombinant antigen as positive control)

• Signal Development:

  • Employ enhanced chemiluminescence for detection

  • Consider longer exposure times (1-5 minutes) for optimal visualization

This methodology aligns with approaches seen in similar membrane protein studies, such as those conducted for SPCA1 antibodies in neural cell cultures .

How can I validate SPAC15A10.06 antibody specificity in my experimental system?

Rigorous validation of antibody specificity is essential, particularly for less characterized targets like SPAC15A10.06. A comprehensive validation approach includes:

• Positive and Negative Controls:

  • Use the supplied recombinant antigen (200μg) as a positive control

  • Use pre-immune serum (1ml provided) as a negative control

  • Include samples from knockout/knockdown cells if available

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunogen peptide

  • Run parallel assays with blocked and unblocked antibody

  • Signal elimination/reduction confirms specificity

• Cross-Reactivity Assessment:

  • Test the antibody against related proteins within the CPA1 transporter family

  • Evaluate species cross-reactivity if working with organisms beyond S. pombe

• Mass Spectrometry Validation:

  • Immunoprecipitate the target protein using the antibody

  • Confirm identity through mass spectrometry analysis

  • Map detected peptides to the SPAC15A10.06 sequence

• Multiple Detection Methods:

  • Confirm results across different techniques (Western blot, ELISA, immunofluorescence)

  • Look for consistent patterns of detection across methods

This multi-faceted approach, similar to validation methods employed for other membrane transporters, provides robust confirmation of antibody specificity .

How can computational approaches enhance SPAC15A10.06 antibody-antigen interaction studies?

Computational methodologies can significantly advance SPAC15A10.06 antibody research through:

• Structure Prediction and Modeling:

  • Homology modeling can predict the 3D structure of SPAC15A10.06 protein using known structures of related Na+/H+ antiporters

  • RosettaAntibody can be employed to model the antibody structure when crystallographic data is unavailable

  • Integration of these models can predict epitope-paratope interactions

• Antibody-Antigen Docking:

  • Two-step docking approach (global and local) can identify potential binding conformations

  • SnugDock can refine binding poses by allowing flexibility of interfacial side chains and CDR loops

  • Energy minimization with RosettaRelax improves conformational accuracy

• Hotspot Identification:

  • Computational alanine scanning can identify critical residues in the antibody-antigen interface

  • These hotspots guide rational antibody engineering for improved specificity

• Affinity Maturation In Silico:

  • Computational protocols can predict mutations to enhance binding affinity

  • Rosetta scoring functions evaluate stability and affinity improvements of designed variants

  • Molecular dynamics simulations can reveal allosteric effects during antibody-antigen recognition

This computational pipeline has demonstrated success in enhancing antibody properties across multiple studies, including redesigned antibodies with improved affinity and stability .

What are the considerations for using SPAC15A10.06 antibody in cross-species studies?

When extending SPAC15A10.06 antibody use beyond S. pombe, researchers should evaluate:

• Sequence Conservation Analysis:

  • Perform multiple sequence alignments of SPAC15A10.06 orthologs across species

  • Calculate percent identity and similarity in epitope regions

  • Predict cross-reactivity based on conservation scores

• Epitope Mapping:

  • Determine the specific epitope recognized by the antibody

  • Evaluate conservation of this epitope in target species

  • Consider synthetic peptide arrays to confirm cross-reactivity

• Validation in Target Species:

  • Perform Western blot with positive and negative controls from target species

  • Include competition assays with recombinant proteins from both S. pombe and target species

  • Validate with orthogonal detection methods

• Experimental Design Adjustments:

  • Optimize antibody concentration for cross-species applications

  • Adjust incubation conditions (time, temperature, buffer composition)

  • Consider post-translational modifications that might differ between species

• Data Interpretation:

  • Exercise caution when interpreting cross-species results

  • Account for potential differences in protein expression, localization, and function

  • Corroborate findings with species-specific tools when available

These considerations are particularly important as the current SPAC15A10.06 antibody is designed specifically against the yeast protein, with species reactivity listed primarily for yeast .

What strategies can address non-specific binding when using SPAC15A10.06 antibody?

Non-specific binding is a common challenge with antibodies targeting membrane proteins like SPAC15A10.06. Effective mitigation strategies include:

• Blocking Optimization:

  • Test different blocking agents (BSA, casein, normal serum from antibody host species)

  • Increase blocking time (2-3 hours at room temperature)

  • Add 0.1-0.5% Tween-20 or Triton X-100 to reduce hydrophobic interactions

• Antibody Dilution Optimization:

  • Perform titration experiments (1:500 to 1:5000) to identify optimal concentration

  • Consider two-step incubation with increasing dilutions

  • Reduce primary antibody concentration if background is high

• Buffer Modifications:

  • Increase salt concentration (150-500mM NaCl) to reduce electrostatic interactions

  • Add 5% polyethylene glycol to reduce non-specific adsorption

  • Consider mild detergents specific for membrane protein applications

• Wash Protocol Enhancement:

  • Increase wash duration and number of washes

  • Use buffers with higher detergent concentration (0.1-0.3% Tween-20)

  • Consider adding 0.5M urea in wash buffer for stubborn non-specific binding

• Pre-adsorption Strategy:

  • Pre-incubate antibody with extracts from null mutants or unrelated species

  • Use the pre-immune serum provided with the antibody to identify non-specific signals

  • Consider immunodepletion against known cross-reactive proteins

These approaches, combined with proper controls, can significantly improve signal-to-noise ratio in SPAC15A10.06 detection assays.

How can I optimize SPAC15A10.06 antibody for immunofluorescence applications?

While the current product information specifically lists ELISA and Western blot as validated applications , researchers interested in immunofluorescence applications should consider:

• Fixation Method Selection:

  • For membrane proteins like SPAC15A10.06, compare 4% paraformaldehyde (preserves structure) with methanol (enhances accessibility)

  • Test mild permeabilization with 0.1-0.2% saponin to preserve membrane integrity

  • Consider short (5-10 min) fixation times to prevent epitope masking

• Antigen Retrieval Optimization:

  • Test heat-induced epitope retrieval (citrate buffer, pH 6.0, 95°C for 10-20 min)

  • Compare with proteolytic digestion methods (0.01-0.1% proteinase K for 5-10 min)

  • Consider detergent-based methods (0.5% Triton X-100 for 10-15 min)

• Signal Amplification Approaches:

  • Implement tyramide signal amplification for low-abundance targets

  • Consider secondary antibody with higher fluorophore conjugation ratio

  • Evaluate biotin-streptavidin amplification systems

• Imaging Optimization:

  • Use confocal microscopy with appropriate Z-stack sampling

  • Implement deconvolution algorithms to enhance signal resolution

  • Consider super-resolution techniques for detailed localization

• Validation Controls:

  • Co-localize with known Golgi markers (e.g., GM130, TGN46)

  • Perform competition assays with recombinant antigen

  • Compare staining patterns with GFP-tagged SPAC15A10.06 expression

Researchers should conduct preliminary validation studies before applying this antibody to immunofluorescence applications given its current validation status.

How can SPAC15A10.06 antibody contribute to understanding membrane protein trafficking dynamics?

The SPAC15A10.06 antibody represents a valuable tool for investigating fundamental questions in membrane protein biology:

• Golgi Transport Mechanisms:

  • Track SPAC15A10.06 localization during cell cycle progression

  • Investigate co-transport with other membrane proteins

  • Analyze response to ion gradient disruptions

• Stress Response Studies:

  • Monitor SPAC15A10.06 expression and localization under ionic stress conditions

  • Evaluate changes in response to pH fluctuations

  • Assess protein stability during ER and Golgi stress

• Interactome Analysis:

  • Use the antibody for co-immunoprecipitation to identify interaction partners

  • Validate interactions through proximity ligation assays

  • Map the broader network of CPA1 transporter family interactions

• Evolutionary Conservation:

  • Compare localization patterns across species with conserved antiporters

  • Investigate functional conservation through complementation studies

  • Trace evolutionary adaptations in membrane transport mechanisms

• Disease Models:

  • Explore potential roles in pathologies related to ion transport dysfunction

  • Investigate connections to pH-dependent protein misfolding diseases

  • Assess implications for drug development targeting membrane transporters

These research directions would benefit from combining antibody-based detection with genetic approaches, such as CRISPR-Cas9 modification of the endogenous protein.

What are the considerations for developing improved versions of SPAC15A10.06 antibodies?

For researchers interested in antibody engineering to enhance SPAC15A10.06 detection, consider:

• Epitope-Focused Design:

  • Target highly specific, conserved epitopes within SPAC15A10.06

  • Use computational alanine scanning to identify optimal antigen regions

  • Consider multiple epitopes to develop complementary antibodies

• Affinity Maturation Approaches:

  • Apply computational affinity maturation protocols to enhance binding

  • Implement directed evolution methods like phage display

  • Use Rosetta-based scoring functions to evaluate theoretical improvements

• Format Optimization:

  • Develop recombinant antibody formats (scFv, Fab) for improved tissue penetration

  • Engineer antibody fragments for specific applications (intrabodies, detection)

  • Consider fusion proteins for specialized detection methods

• Cross-Reactivity Engineering:

  • Design antibodies targeting conserved epitopes for cross-species applications

  • Implement negative selection strategies against homologous proteins

  • Balance specificity with broader utility through rational design

• Application-Specific Modifications:

  • Develop directly conjugated versions for immunofluorescence

  • Engineer protease-resistant variants for harsh application conditions

  • Consider humanized versions for potential therapeutic applications

The implementation of in silico antibody design protocols like IsAb can significantly accelerate these engineering efforts and reduce experimental costs .